The existence of tumor-initiating cancer stem cells (CSC) has been shown in a variety of solid tumors (e.g., breast, prostate, glioblastoma, liver). However, these CSC have highly variable antigenic and functional properties even when derived from the same tumor. These observations highlight a cardinal problem in CSC biology, namely, the heterogeneity of the CSC. Different mechanisms could explain the origin and heterogeneity of CSC such as (i) differentiation arrest (stem cells), (ii) dedifferentiation (mature cells) and (iii) transdifferentiation (bone marrow stem cells). It is conceivable that all 3 mechanisms may be corrupted by oncogenic events, resulting in an assortment of CSC and explaining their heterogeneity. Defining and characterizing this heterogeneity is of vital importance for understanding CSC biology, and for effective therapeutic translation. Our most recent results in this project include: (1)Human hepatocellular carcinoma (HCC) is a heterogeneous disease of distinct clinical subgroups. A principal source of tumor heterogeneity may be cell type of origin, which in liver includes hepatocyte or adult stem/progenitor cells. To address this issue, we investigated the molecular mechanisms underlying the fate of the enzyme-altered preneoplastic lesions in the resistant hepatocyte (RH) model. Sixty samples classified as focal lesions, adenoma, and early and advanced HCCs were microdissected after morphological and immunohistochemical evaluation and subjected to global gene expression profiling. The analysis of progression of the persistent glutathione S-transferase (GSTP)(+) focal lesions to fully developed HCC showed that approximately 50% of persistent nodules and all HCCs expressed cytokeratin 19 (CK19), whereas 14% of remodeling nodules were CK19(+). Unsupervised hierarchical clustering of the expression profiles also grouped the samples according to CK19 expression. Furthermore, supervised analysis using the differentially expressed genes in each cluster combined with gene connectivity tools identified 1308 unique genes and a predominance of the AP-1/JUN network in the CK19(+) lesions. In contrast, the CK19-negative cluster exhibited only limited molecular changes (156 differentially expressed genes versus normal liver) consistent with remodeling toward differentiated phenotype. Finally, comparative functional genomics showed a stringent clustering of CK19(+) early lesions and advanced HCCs with human HCCs characterized by poor prognosis. Furthermore, the CK19-associated gene expression signature accurately predicted patient survival (P &lt;0.009) and tumor recurrence (P &lt;0.006). Conclusion: Our data establish CK19 as a prognostic marker of early neoplastic lesions and strongly suggest the progenitor derivation of HCC in the rat RH model. The capacity of CK19-associated gene signatures to stratify HCC patients according to clinical prognosis indicates the usefulness of the RH model for studies of stem/progenitor-derived HCC.(2)The ability to retain DNA labels over time is a property proposed to be associated with adult stem cells. Recently, label retaining cells (LRC) were indentified in cancer. LRC were suggested to be the result of either slow-cycling or asymmetric-cell-division with nonrandom-chromosomal-cosegregation (ACD-NRCC). ACD-NRCC is proposed to segregate the older template DNA strands into daughter stem cells and newly synthesized DNA into daughter cells destined for differentiation. The existence of cells undergoing ACD-NRCC and the stem-like nature of LRC remain controversial. Currently, to detect LRC and ACD-NRCC, cells need to undergo fixation. Therefore, testing the stem-cell nature and other functional traits of LRC and cells undergoing ACD-NRCC has been limited. Here, we show a method for labeling DNA with single and dual-color nucleotides in live human liver cancer cells avoiding the need for fixation. We describe a novel methodology for both the isolation of live LRC and cells undergoing ACD-NRCC via fluorescence-activated cell sorting with confocal microscopy validation. This has the potential to be a powerful adjunct to stem-cell and cancer research.(3)Current evidence suggests that hepatocellular carcinoma (HCC), the most common adult primary liver cancer, comprises biologically distinct subgroups including the recently identified hepatic progenitor cell- and cholangiocarcinoma (CC)-like subtypes. These observations support the notion that HCCs can be derived from different cells within the lineage from adult liver stem cell to hepatocyte. We addressed two questions: (1) does the transforming potential of oncogenes depend on the stage of hepatocytic differentiation and (2) is the tumor phenotype determined by the stage of differentiation at which transformation occurs. Fetal (ED16.5) hepatocytes were purified by magnetic sorting and adult (3-month) hepatocytes were isolated by collagenase perfusion from C57BL/6 mice. Cells were transduced with lentiviral vectors carrying oncogenic H-Ras and SV40 large T (LT) together with GFP and mCherry fluorescent reporters. Double positive cells were sorted and injected into the spleen of NOD/SCID mice. Tumors larger than 3 mm derived from the transduced fetal (n=20) and adult hepatocytes (n=20) were macrodissected and subjected to histopathology evaluation, immunohistochemistry and transcriptome profiling. Cancer stem cell traits were examined by FACS analysis of lineage specific and cancer stem cell associated markers, side population and tumor sphere formation assays using cell lines established from fetal and adult hepatocyte-derived tumors. Both fetal and adult hepatocytes co-transduced with oncogenic H-Ras/SV40LT produced liver tumors with similar growth characteristics and gave rise to lung metastases with 80% (8/10) and 63% (5/8) frequency, respectively. Histologically, fetal and adult hepatocyte-derived tumors were moderately-poorly differentiated HCCs with CC-like phenotype. All tumor cell lines were 100% positive for CK18, CK19, EpCam, A6, CD44, CD49f and CD29 and contained SP fraction (1-2%) as measured by FACS analysis regardless of the cell origin. CD133 was expressed at lower and variable levels in fetal (26.07.2%) and adult (46.19.1%) tumor cell lines. Both fetal and adult tumor cells possessed a sphere forming capacity which was maintained within 5 sphere generations. Unsupervised hierarchical clustering separated the fetal and adult tumors after log2 transformation by using corresponding normal samples as reference. Our results suggest that the activation of certain oncogenic pathways may have more significant effect on the tumor phenotype than the stage of hepatocytic differentiation at which the transformation occurs. Oncogenic H-Ras and SV40LT are capable of inducing a cancer stem cell-like state in mature hepatocytes that displays characteristics of hepatic stem cells.